Moving heat blocks for amplification of nucleic acids

ABSTRACT

An apparatus for thermally processing reaction material containing nucleic acid is provided. A reaction material is contained in reactors. The apparatus includes a reactor holder for statically holding the reactors; at least two heating means each being maintainable at a user specifiable temperature; and a transport means for positioning the heating means to make a contact with the reactors one at a time for specified duration. The positioning is conductable once or over a plurality of times for thermally processing the reactors between a plurality of temperatures.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the Continuation In Part Application of International Application PCT/SG2017/050286, filed on 6 Jun. 2017, which is based upon and claims priority to American Patent Application No. 62/348,155, filed on 10 Jun. 2016, Singaporean Patent Application No. 10201700260X, filed on 12 Jan. 2017, and Singaporean Patent Application No. 10201702663P, filed on 31 Mar. 2017, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the field of amplification of nucleic acids for analyses.

BACKGROUND

Amplification of nucleic acids such as for polymerase chain reaction (PCR), isothermal amplification, and primer extension, is increasingly important to genetic engineering, molecular biology, food safety, environmental monitoring, medicine for analyses and diagnosis. A large number of biological researchers use DNA amplification techniques such as PCR in their work on nucleic acid analyses, due to its high sensitivity and specificity. The DNA amplification such as PCR is conducted in a PCR module where the nucleic acid reaction material undergoes thermal processing such as thermal cycling for DNA amplification. The optical detection may be carried during and/or after the thermal cycling. Researchers have been constantly striving to increase the speed of thermal cycling.

The time duration of a PCR is typically in the order of an hour, primarily due to a time-consuming PCR thermal cycling process that requires the heating device to be cyclically regulated to attain target temperatures for heating and cooling reactors containing the sample for DNA denaturation, annealing and extension.

In order to speed up the process of thermal cycling, multiple heating devices are also known to be used, where each device can be set to a specified temperature as required for the thermal cycling. The heating devices commonly used are water baths maintained at specified temperatures. The reactors can then be transported between these devices for the thermal cycling. However, using the liquid baths have several disadvantages like loss of liquid due to evaporation that needs to be topped up as required, leakages and the kind. The liquid having gone through one or more rounds of thermal cycling may also progressively change its volume and properties thereby affecting the time and temperature calibration of the apparatus, particularly when automated.

As disclosed under the U.S. Pat. No. 9,061,285 B2, a faster speed of the PCR may be achieved when the thermal cycling apparatus moves a biochip reactor between two heating blocks whose temperatures are set at the target temperatures as required for nucleic acid amplification reactions. This apparatus works for a PCR biochip only in which the chip has a flat bottom surface to be in contact with the flat heat block. However, the biochip is far less cost effective and user-friendly than the PCR tubes and wellplates. As compared to a biochip, the conventional PCR tubes and wellplates are also advantageous in their ability to interface with standard liquid handling robots and multi-channel pipettes, allow easy loading and aspiring of liquid in the wells, either manually and robotically.

The present invention provides a thermal cycling apparatus with higher efficiency of the thermal processing of reactors such as the conventional PCR tubes, PCR wellplates and biochip. The invention allows an improved architecture of the apparatus with easier maintenance and higher precision for imaging.

SUMMARY

Unless specified otherwise, the term “comprising” and “comprise” and grammatical variants thereof, are intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, unrecited elements. The terminologies ‘first heating block’, ‘second heating block’ . . . ‘sixth heating block’ do not constitute the corresponding number of blocks in a sequence but merely are names for ease of identification with respect to the purpose they serve. These baths may not necessarily represent separate physical entities as some of them may be shareable. The term ‘thermal processing’ includes: a) thermal cycling, and optionally includes: b) thermal process steps before and/or after thermal cycling. The term ‘thermal profile’ refers to the temperature-time variation of the reactor(s) during a) alone or during a) with b).

Apparatus for thermal processing reaction material containing nucleic acid is provided, the reaction material being contained in reactor(s) and the reactor(s) being in any form such as tube(s) or wellplate(s) or chip(s) or cartridge(s). The apparatus comprises a reactor holder for statically holding the reactor(s); at least two heating blocks each being maintainable at a specified temperature; and transport means for positioning the at least two heating blocks to make a contact with the reactor(s) one at a time for specified duration, the positioning being conductable once or over a plurality of times for thermal processing the reactor(s) between a plurality of user specifiable temperatures. Herein, faster speed of the thermal processing is achieved with the multiple heating blocks each set at a specified temperature. The heat blocks advantageously eliminate the disadvantages of using the conventional liquid baths that suffer from leakages, spillages and loss of liquid due to evaporation hence requiring frequent top-up. The liquid vapors may also contaminate the surrounding regions of the apparatus. The liquid having gone through one or more rounds of thermal cycling may also progressively change its properties, thereby affecting the time and temperature calibration of the apparatus. The apparatus with the moving heating blocks enables the reactor(s) to remain static during the thermal processing, therefore also enables the optical module for optical detection of the reaction material in the reactor(s) to remain static during the process. This advantageously avoids potential misalignment that could be caused by any relative movement between the optical module and the reactor(s). Additionally, tight contacts between the reactor(s) and the heating blocks for a faster heat exchange can be easily attained by pushing the heating block against the reactor(s) by the electromechanical modules for moving the heating blocks. Advantageously, such electromechanical modules can be accommodated towards the bottom of the apparatus thereby allowing better space with lesser mechanical mass towards the upper part of the apparatus where the delicate reactor(s) and the optical module are accommodated. The space also facilitates easy access to the reactor(s) by other modules like sample preparation module using liquid dispensing pipettes and reactor sealing module using various mechanisms in the art. The apparatus is thus robust, light weight, easy to use and can handle the cheap and commercially available disposable reactors for the reaction material to avoid cross contamination from one reactor to the next. The apparatus is operable at a high speed and at affordable cost, without using complex and expensive components or consumables. This invention provides a great positive impact on biological analysis.

According to an embodiment, the transport means provides linear displacement for the heating blocks, such that the selected heating block may be positioned under the reactor(s), thereafter moved upwards to make contact with the reactor(s) for specified duration and thereafter moved downwards to break the contact and proceed with linear displacement of another heating block. This is a simple arrangement that works well when two blocks are used for thermal cycling.

According to another embodiment, the transport means further provides rotational movements for moving the heating blocks in a circular path, the rotational movement being along a common axis for the at least two heating blocks. The movement of the heating blocks in the circular path minimizes the footprint especially when there are more than two heating blocks. The circular path may be along a vertical plane for a smaller footprint as compared with an embodiment when the circular path is along a horizontal plane.

According to an embodiment, the contact between the reactor(s) with at least one of the heating blocks is made when the reactor(s) gets inserted into cavities in the at least one of the heating blocks, the shape of the cavities being substantially conformal with at least the lower portions of the reactor(s). This feature provides tight and uniform contact of the reactor(s) with the heating blocks for speedy heat exchange between the heating blocks and the reactor(s), which is desirable both for the quality control of the thermal processing and also for reducing the processing tune.

According to an embodiment, the heating blocks comprise thermally conductive elastomeric material such as silicone. Since silicone can be deformed, a tight fit of the reactor(s) in the cavities may be obtained with the cavities being slightly smaller than the reactor(s) when made of glass or metal. According to an alternate embodiment, the heating block comprises porous metal wherein the voids are filled with a thermally conductive liquid. This feature allows a tight fit between the cavities and the reactor(s) by using cavities which are slightly undersized than the glass or metallic reactor(s), such that slight deformation of the cavities occur causing the thermally conductive liquid to fill up any gap in between the cavities and the reactor(s).

According to an embodiment, each of the heating blocks comprises: a first portion that is coupled to the transport means; and a second portion that can be removably coupled to the first portion, the second portion accommodating the cavities. This feature helps the user to easily replace the second portion to suit the available reactor pitch and design of the reactors in the form of tubes or well plates. This feature allows the first portion that is coupled to the transport means to remain undisturbed in the interest of maintaining precision of the positioning the cavities to make the contacts with the reactor(s).

According to an embodiment, each heating block comprises at least one injector means such that in operation, the injector means provides a pressure on the reactor(s) to release the contact, before the reactor(s) separates out of the heating block. This feature is useful particularly when the contact between the reactor(s) and the cavity is maintained as a tight fit to improve the heat exchange. The injector means may access the reactor(s) via a through hole in the heating block which provides an effective way to release the contact in a compact arrangement for the injector means.

At least one of the heating blocks may be a solid metallic block. Advantageously, the metallic block is easier to fabricate. According to another embodiment, at least one of the heating blocks is a hollow metallic block that can accommodate heating medium such as liquid or mica. This is advantageous if the liquid or mica makes the block lighter than when the block is a solid metallic block. Lighter blocks are easier to handle by the transport means and facilitate lighter weight of the apparatus. Lighter blocks also allow lesser power consumption by the transport means.

According to an embodiment, the apparatus further comprises fluorescence imaging means that allows light to access the nucleic acid when the reactor(s) is/are in contact with any of the heating block or is/are in air outside the heating block as desired by the user.

According to an embodiment, at least a portion of at least one of the heating blocks is transparent to light for fluorescent imaging of the reaction material while the reactor(s) is/are in the contact with the at least one of the heating blocks.

The heating block may be at least partially made of a transparent material that is in contact with a transparent electrical conductive layer for allowing resistive heating. The transparent material may be glass. The transparent electrical conductive material may be ITO (Indium Tin Oxide). The transparency is useful for fluorescent imaging of the reaction material.

The apparatus may comprise a temperature sensor means or a time sensor means to determine the specified duration. The temperature sensor means may be located to sense the real-time temperature in a sample reactor containing the reaction material. This closely simulates the reactors undergoing the thermal processing. The apparatus may further comprise a calibration means for user calibrating the time sensor means. This feature is helpful particularly (though not exclusively) when the target temperature is lower than the user specifiable temperature or when the target temperature is higher than the user specifiable temperature in at least one of the heating blocks.

According to an embodiment, the at least two heating blocks comprise: a first heating block where the reactor(s) is/are allowed to attain a predetermined high target temperature T_(HT), wherein the Ti is in the region 85-99 degree Celsius for denaturation of the nucleic acid; and a second heating block where the reactor(s) is/are allowed to attain a predetermined low target temperature T_(LT), wherein the T_(LT) is in the region 45-75 degree Celsius for annealing of primers or probes onto nucleic acid or for primer extension for thermal cycling the reactor(s) to attain polymerase chain reaction (PCR) amplification or primer extension. These are typical temperature ranges for PCR thermal cycling. According to another embodiment, the apparatus further comprises a third heating block where the reactor(s) is/are allowed to attain a predetermined medium target temperature T_(MT), wherein the T_(MT) is for annealing of primers or probes onto nucleic acid. The apparatus may further comprise a fourth heating block where the reactor(s) is/are allowed to attain a predetermined medium target temperature T_(MT), wherein the T_(MT) is for extension of primers on nucleic acid. This feature further advantageously utilizes the inventive concept. The T_(MT) may be user settable to T_(LT) for achieving a desired thermal profile.

The apparatus may further comprise a fifth heating block where the reactor(s) is/are allowed to attain a temperature TAP to allow an additional process for the reactor(s) before thermal cycling, the additional process being one from the group consisting: reverse transcription-polymerase chain reaction (RT-PCR), hot start process and isothermal amplification reaction. The apparatus may further comprise a sixth heating block that can be progressively heated while conducting melt curve analysis after thermal cycling. The fifth and the sixth blocks allow the thermal cycling to be integrated with the previous and the following steps respectively while advantageously using the inventive concept.

According to an embodiment, at least one of the heating block is further capable of providing temperatures with programmable ramp up or ramp down characteristics for providing user flexibility on the thermal profiles. According to an embodiment, at least one of the heating block is further capable of providing plurality of the user specifiable temperatures for use before or after the thermal processing such as for reverse transcription-polymerase chain reaction (RT-PCR), isothermal amplification reaction, DNA melting analysis and the kind. The specified duration may be dependent on a target temperature to be achieved by the reactor(s) when in the contact, irrespective of any offset in the temperatures for the blocks so that the thermal processing is not adversely affected.

According to an embodiment, the apparatus further comprises a sealing means for sealing the reactor(s) before the thermal processing to prevent the reaction material from vaporizing during thermal processing. During the sealing, the reactors may be positioned over at least one of the heating block, so that after sealing the reactors need not have to be moved over the heating block during the next step of thermal cycling. This saves time and requires no further operating mechanism thereby further saving on the mass and the footprint of the apparatus. According to yet another embodiment, the apparatus further comprises a sample preparation means for preparing the reaction material and loading into the reactor(s) before the thermal processing. Integrating the whole process in a single apparatus helps in automating the processing line and also overcomes the requirement of providing controlled ambience and trained personnel for conducting the process of sample preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following drawings, same reference numbers generally refer to the same parts throughout. The drawings are not to scale, instead the emphasis is on describing the concept.

FIGS. 1(a) to (e) presents elevation cross-sectional views of the apparatus with two heating blocks changing their positions by a linear-rotary motion stage during thermal processing, as according to an embodiment of the invention.

FIGS. 2(a) to (f) presents elevation cross-sectional views of the apparatus with three heating blocks changing their positions by a linear-rotary motion stage, as according to an embodiment of the invention.

FIGS. 3(a) to (f) presents elevation cross-sectional views of the apparatus with six heating blocks changing their positions by a linear-rotary motion stage, as according to an embodiment of the invention.

FIGS. 4(a) to (d) presents elevation cross-sectional views of the apparatus with two heating blocks changing their positions by a linear-linear motion stage, as according to an embodiment of the invention.

FIG. 5 is a planar view of the apparatus with four heating blocks changing their positions relative to the reactor(s) in a horizontal and circular path, as according to an embodiment of the invention.

FIG. 6 is an isometric view of an embodiment of the apparatus including the sealing means and the sample preparation means as according to an embodiment of the invention.

FIGS. 7 (a) to (c) show elevation cross-sectional views of the reactor when in contact with the cavity, according to an embodiment of the invention.

FIGS. 8 (a) to (c) in elevation cross-sectional views illustrate an embodiment of the invention using injector pins to release the tight fit between the reactors and the cavities.

FIGS. 9 (a) and (b) in a cross-sectional view illustrates an embodiment of the invention wherein the heating block is split into two portions.

FIG. 10 in elevation cross-sectional view illustrates an embodiment of the invention wherein the heating block includes a bath and a bath medium.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following description presents several preferred embodiments of the present invention in sufficient detail such that those skilled in the art can make and use the invention.

According to a first embodiment 100, FIGS. 1(a) to (e) illustrate the movement of a first heating block 10 and a second heating block 12 which are maintained at two different temperatures during the thermal cycling of the reaction material within the reactors 5. The reactors 5 thus need to cycle between the two heating blocks 10, 12 several times as predetermined. The heating blocks 10, 12 change their positions by a rotary motion stage 22 and a linear motion stage 24, as according to an embodiment of the invention. The rotary motion stage 22 enables the heating blocks 10 and 12 to rotate along a vertical plane and about a common axis. As shown at FIG. 1(a), the reactors 5 presumably containing the reaction material are capped by the cap strip 2. The reactors 5 are held in a static position by the reactor holder 4. The first heating block 10 is placed in the active position where the reactors 5 make contact with the first heating block 10 by each inserting into the cavities 15 of the first heating block 10. The second heating block 12 is shown in a passive position. After a predetermined time as shown at FIG. 1(b), the linear motion stage 24 linearly displaces the first heating block 10 downwards as shown by the dashed block arrow and the rotary motion stage 22 rotates as shown by the curved arrow such that the second heating block 12 is positioned under the reactors 5. At FIG. 1(c), the linear motion stage 24 linearly displaces the second heating block 12 upwards to make contact with the reactors 5 and remain in this active position for another predetermined time. As shown at FIG. 1(d), the linear motion stage 24 linearly displaces the second heating block downwards as shown by the dashed block arrow and the rotary motion stage 22 is rotated as shown by the curved arrow such that the first heating block 10 is positioned under the reactors 5. As shown at FIG. 1(e), the linear motion stage 24 again linearly displaces the first heating block 10 upwards to the active position to make contact with the reactors 5 as at FIG. 1(a). This sequence may be repeated several times as required under the process of thermal processing. The temperatures of the two heating blocks 10, 12 are maintained at predetermined temperatures as required.

According to a second embodiment 200, FIGS. 2(a) to (f) illustrate the movement of the first heating block 10, the second heating block 12 and a third heating block 14 which are maintained at three different temperatures during the thermal cycling of the reaction material within the reactors 5. The operations of the motion stages 22, 24 are similar to that described under FIG. 1, but with three heating blocks 10, 12, 14 instead of two 10, 12. The sequence of positioning the heating blocks 10, 12, 14 in the active position may be altered as required for the process of thermal cycling. The sequence shown in the figures is self-explanatory.

As in FIG. 2, according to an embodiment of conducting thermal cycling for PCR amplification, the first heating block 10 is maintainable at a temperature T_(H) which is higher than a target temperature T_(HT). For example, T_(H)=150 degrees Celsius and T_(HT)=95 degrees Celsius. The first heating block 10 is displaced downwards (or reactor 5 lifts up from the heating block 10) once the reactors 5 have attained T_(HT) and the second heating block 12 is positioned under the reactors 5 as in FIG. 2(c). The second heating block 12 is maintainable at a temperature T_(L) which is lower than a target temperature T_(LT). For example, T_(L)=10 degrees Celsius and T_(LT)=60 degrees Celsius. The second heating block 12 is displaced downwards (or reactor 5 lifts up from the heating block 12) once the reactors 5 have attained T_(LT) and the third heating block 14 is positioned under the reactors 5 as in FIG. 2(e). The third heating block 14 is maintained at a temperature T_(L)=60 degrees Celsius for temperature stabilization for a period of time, for annealing and primer extension. FIG. 2 (f) shows the start of the next thermal cycle. The temperature offset between T_(H) and T_(HT), and between T_(L) and T_(LT) respectively increase the heating rate and the cooling rate of the reactors 5. The T_(HT) is for the denaturation of the nucleic acid, and the T_(LT) is for annealing of primers or probes onto nucleic acid and for primer extension. A fluorescent imaging of reactor 5 may be conducted when the reactor 5 is in the third heating block 14.

According to a third embodiment 300, FIGS. 3(a) to (f) illustrate the movement of six heating blocks 10, 12, 14, 16, 18, 20, each being maintained at a different temperature during the thermal processing of the reaction material within the reactors 5. The operations of the motion stages 22, 24 are similar to that described under FIG. 1, but with six heating blocks 10, 12, 14, 16, 18, 20. The sequence of positioning the heating blocks 10, 12, 14, 16, 18, 20 in the active position may be altered as required for the thermal processing. The sequence shown in the figures is self explanatory. The heating blocks 10 may be maintained at temperatures for additional process before thermal cycling like reverse transcription-polymerase chain reaction (RT-PCR), hot start process and isothermal amplification reaction. The heating blocks 12, 14, 16 may be respectively maintained at temperatures for denaturation, annealing and extension. The heating blocks 18 and 20 may be maintained at temperatures for stabilization and imaging.

As in FIG. 3, according to an embodiment of conducting thermal cycling for PCR amplification, the first heating block 10 is maintainable at the temperature T_(H) which is higher than the target temperature T_(HT). For example, T_(H)=150 degrees Celsius and T_(HT)=95 degrees Celsius. The first heating block 10 is displaced downwards (or reactor 5 lifts up from the heating block 10) once the reactors 5 have attained T_(HT) and the second heating block 12 that is maintained at T_(HT) is positioned under the reactors 5. The second heating block 12 provides temperature stabilization at T_(HT). The third heating block 14 is maintainable at the temperature T_(L) which is lower than the target temperature T_(LT). For example, T_(L)=10 degrees Celsius and T_(LT)=60 degrees Celsius. The third heating block 14 is displaced downwards (or reactor 5 lifts up from the heating block 14) once the reactors 5 have attained T_(LT) and the fourth heating block 16 is positioned under the reactors 5. The fourth heating block 16 is maintained at the temperature T_(LT)=60 degrees Celsius for temperature stabilization. The fourth heating block 16 is displaced downwards (or reactor 5 lifts up from the heating block 16) once the reactors 5 have stabilized at T_(LT) and the fifth heating block 18 is positioned under the reactors 5. The fifth heating block 18 is maintained at the temperature T_(M)=80 degrees Celsius which is higher than T_(MT)=72 degree Celsius. The fifth heating block 18 is displaced downwards (or reactor 5 lifts up from the heat block 18) once the reactors 5 have attained T_(MT) and the sixth heating block 20 is positioned under the reactors 5 for stabilization at T_(MT). The temperature offset between T_(H) and T_(HT), between T_(L) and T_(LT) and between T_(M) and T_(MT) respectively increase the heating rate and the cooling rate of the reactors 5. The T_(HT) is for the denaturation of the nucleic acid, and the T_(LT) is for annealing of primers or probes onto nucleic acid or for primer extension. A fluorescent imaging may be conducted at this third heating block 14. T_(MT) is for stabilization.

According to a fourth embodiment 400, FIGS. 4(a) to (d) illustrate the linear movement of two heating blocks 10, 12, being maintained at two different temperatures during the thermal processing of the reaction material within the reactors 5. In this embodiment, both the heating blocks 10 and 12 have a common platform 40. The linear motion stage 24 (not shown) provides linear displacement of the heating blocks 10, 12 via the common platform 40. At FIG. 4(a), the first heating block 10 is in the active position. At FIG. 4(b), the heating blocks 10, 12 are displaced downwards. At FIG. 4(e), the heating blocks 10, 12 move towards the left as shown by the dashed block arrow. At FIG. 4(d), the heating blocks 10, 12 are linearly displaced upwards to place the second block 12 in the active position. According to other embodiments, more number of heating blocks may be used.

According to a fifth embodiment 500, FIG. 5 in a plan view illustrates the movement of four heating blocks 10, 12, 14, 16 in a horizontal plane and about a common axis. Each of the heating blocks 10, 12, 14, 16 is maintainable at a different temperature during the thermal processing of the reaction material within the reactors 5. The operation of the motion stages 22, 24 (not shown) is similar to that described under FIG. 1, but with the four heating blocks 10, 12, 14, 16 and rotating in the circular path in the horizontal plane instead of the vertical plane. The sequence of positioning the heating blocks 10, 12, 14, 16 in the active position may be altered as required for the process of thermal processing.

As in FIG. 5, according to an embodiment of conducting thermal cycling for PCR amplification, similar temperature offset is maintained, where the first heating block 10 is maintainable at the temperature T_(H) which is higher than the target temperature T_(HT). For example, T_(H)=150 degrees Celsius and T_(HT)=95 degrees Celsius. The first heating block 10 is displaced downwards (or reactor 5 lifts up from the heating block 10) once the reactors 5 have attained T_(HT) and the second heating block 12 that is maintained at T_(HT) is positioned under the reactors 5. The second heating block 12 provides stabilization of the reactor temperature at T_(HT). The third heating block 14 is maintainable at the temperature T_(L) which is lower than the target temperature T_(LT). For example, T_(L)=10 degrees Celsius and T_(LT)=60 degrees Celsius. The third heating block 14 is displaced downwards (or reactor 5 lifts up from the heating block 14) once the reactors 5 have attained T_(LT) and the fourth heating block 16 that is maintained at T_(LT) is positioned under the reactors 5. Then the next thermal cycle starts when the first heating block 10 moves under reactor 5.

The above heating block temperatures T_(H), T_(L), T_(M) and the target temperatures T_(HT), T_(LT), T_(MT) may take other suitable values. The above thermal cycling step involving heating block 12 is optional, i:e once the reactor 5 separates from the heating block 10, the heating block 14 moves in.

FIG. 6 shows the apparatus for amplification of nucleic acid as integrated along with the sealing means 100, the sample preparation means 200 and the fluorescence imaging means or the electrochemical detection means 400. In this embodiment, during the sealing, the reactors are shown to be positioned over at least one of the heating block.

The reactors 5 in the tube form as shown could be made up of glass or metal or plastic. While making the contact with the heating blocks 10, 12, 14, 16, 18, 20, the plastic deforms to some extent to fit into the cavities 15. Thus the size of the cavities 15 may be slightly smaller than those of the reactors 5 to ensure a tight fit for a good contact with the cavities 15. However since the glass or metallic ones do not deform, slight oversize of the cavities 15 need to be used thereby forming a gap 34 as shown in FIG. 7 (a). The gap 34 needs to be maintained to avoid collision during the relative movement between the cavity 15 and the reactor 5. This gap 36 may be occupied with air or preferably a thermally conductive lubricant liquid (not shown) such as oil, lubricant chemicals, water and the kind. The liquid is held in the narrow cavity 15 under surface tension and would not flow out even when the heating block 10 is inverted. This concept may be applicable to plastic and metallic reactors 5 too where the shapes of the cavities 15 are not substantially conformal with the reactors 5. The liquid may be replenished manually or robotically as required. As shown in an embodiment at FIG. 7 (b), the heating block 10 may be made of porous metal which contains a large number of voids among the metal phase typically obtained by bonding metal particles together under high pressure and temperature or by a metal infiltration process. The voids can store the thermally conductive liquid that is able to form a liquid layer over the surface of the cavity 15 by means of surface tension or by pressure exerted on the liquid to move to the surface of the cavity. Thus, the gap 34 may be filled with the thermally conductive liquid seeping out from the void region of the porous metal of the heating block 10, thereby forming a conductive liquid layer to enhance heat transfer between the reactor 5 and the heating block 10. In an alternate embodiment as shown in FIG. 7 (c), the heating blocks 10 may be made of a thermally conductive elastomeric material such as silicone or rubber. Since the silicone can be deformed easily under a small stress, in order to ensure a tight fit the cavities 15 can be slightly smaller than the reactors 5.

FIG. 8 (a) illustrates an embodiment for a case when the reactors 5 are subjected to a tight fit in the cavities 15 for a good thermal contact. When the heating block 10 is linearly displaced downwards for breaking the contact, the tight fit resists the detachment of the reactor 5 from the cavity. In order to address this issue, through-holes 36 are provided in the heating block 10 with injector pins 38 inserted from the bottom side of the heating block 10. As shown in FIG. 8 (b), during the detachment, the injector pins 38 provide a small upward force along the bottom side of the reactors 5 to loosen the contact. Thereafter the heating block 10 starts moving downwards as shown by the dashed arrow. As shown in FIG. 8 (c), both the heating block 10 and the pins 38 move downwards for another heating block to take position and make the contact with the reactors 5. The set of injector pins 38 may be provided with each heating block or may be common for all the heating blocks 10, 12, 14, 16, 18, 20, to insert into the through holes 36 when the heating block is in the active position. Such injector pins 38 may be used also for an embodiment where the heating block 10 is stationary and the reactor 5 in the reactor holder 4 moves upwards for breaking the contact. The injector pins 38 may preferably be provided for each reactor 5. However, having fewer ones may also work. The fewer ones may preferably be distributed over the area of the reactors 5 so as to maintain better uniformity of the upward force provided.

According to an embodiment as shown at FIG. 9a , the heating block 10 is split into a top portion 10 a including the cavities 15 and a bottom portion 10 b that is coupled to the rotary and linear motion stages 22, 24 as described earlier. The top portion 10 a is designed to accommodate the tubular and well-plate reactors 5 with varying sizes and pitches. Hence, depending on the size, shape and pitch of the reactors 5, suitable top portions 10 a can be rigidly fixed over the bottom portion 10 b by the user. FIG. 9b shows the top portion 10 a wherein the size, shape and pitch of the cavities 15 are different from the top portion 10 a of FIG. 9a . In this embodiment, the injector pins 38 are shown though it is not a necessary feature.

As show in FIG. 10, any of the heating blocks described above may be interpreted to comprise a bath 12 a and a bath medium 12 b, both the bath 12 a and the bath medium 12 b being in solid phase. The solid materials for the bath 12 a and bath medium 12 b may be same or different. The size and shape of the cavity 15 are substantially the same as that of the outer surface of the reactor 5 for an efficient heat transfer during the thermal cycling.

It will be understood by those skilled in the art that in the preceding embodiments, the shape of the cavities 15 may preferably be substantially conformal with the lower portions of the reactors 5 such that tight and uniform contact of the reactor(s) 5 with the heating blocks 10, 12, 14, 16, 18, 20 may be established for speedy heat exchange. This is desirable both for the quality control of the thermal processing and for reducing the cycle time. The pitch of the cavities 15 in the heating blocks 10, 12, 14, 16, 18, 20 need to match the pitch of the reactors 5 whether as individual reactors 5 held by the reactor holder 4 or in the form of well plates. During the thermal processing, the positioning of the heating blocks 10, 12, 14, 16, 18, 20 under the reactors 5 needs to be executed with the required precision. Temperature sensors (not shown) are provided with each heating block to monitor and regulate the specified temperature. The temperatures of the different heating blocks 10, 12, 14, 16, 18, 20 may co-relate to the target temperatures for the thermal processing such as for DNA denaturation, annealing and extension. For temperature control in the thermal cycling process, the temperature sensor may be mounted inside each heating block or mounted inside a reactor 5 dedicated for temperature sensing which has a similar heat transfer characteristics as the reactor 5 containing the reaction material. Such a reactor 5 with the temperature sensor is inserted into the cavity of the different heating blocks together with the other reactors 5.

The apparatus may be programmed such that the duration of the contact is dependent on a target temperature to be achieved by the reactor(s) when in the contact. The temperature of any of the heating blocks 10, 12, 14, 16, 18, 20 may be set to be higher than the target temperature to speed up the rate of heating of the reactor(s). Similarly the temperature of any of the heating blocks 10, 12, 14, 16, 18, 20 may be set to be lower than the target temperature to speed up the rate of cooling of the reactor(s). In order to enhance the rate of heating for the reactor 5, the user specifiable temperature in one or more of the heating block(s) 10, 12, 14, 16, 18, 20 may be maintained higher than the target temperature such as the denaturation temperature for the reactor 5. The reactor 5 can remain in the heating block(s) 10, 12, 14, 16, 18, 20 for a specified period of time required for denaturation. Similarly In order to enhance the rate of cooling for the reactor 5, the user specifiable temperature may be maintained lower than the target temperature such as the annealing or extension temperature. The reactor 5 can remain in the heating block(s) 10, 12, 14, 16, 18, 20 for another specified period of time as required for annealing and extension for the reactor 5. For such cases it is preferable to have the tube mounted with a temperature sensor insider the tube. When the temperature sensor inside the reactor 5 senses the temperature reaching the target temperature, the rotary motion stage 22 and the linear motion stage 24 would separate the heating block from the reactor 5 and bring in another heating block to contact the reactor 5. The preceding embodiments show the reactors 5 in the form of tubes and the heating blocks 10, 12, 14, 16, 18, 20 to be provided with cavities 15 to receive the lower side of the reactors 5 when in the active position. However, this is not a limitation. Reactors 5 with other shapes including PCR chips and cartridges may also be used. The PCR chip requires multilayer bonding or glue sealing of plates or films which may frequently cause delamination and leaking during the thermal cycling process. The cartridges have similar problems as well. The conventional PCR tubes and wellplates are molded as a single piece plastic without any bonding required. This provides a more reliable vehicle to contain the biological sample and reagent before, during and after the nucleic acid analyses. Moreover, the PCR chip has a flat surface to be in contact with the heating blocks 10, 12, 14, 16, 18, 20 and such one-sided heating and cooling could generate a thermal gradient across the PCR chip thereby making the temperature inside inaccurate and the temperature control difficult. On the contrary, as described in the preceding embodiments, the reactors 5 in the form of conventional PCR tube or a tube on a PCR wellplate are inserted into the cavities 15 for heating and cooling, and such cavities 15 surround the tube in all directions making the heating and cooling uniform over the domain of the reaction material in the tube. This helps the temperature field to be more uniform inside the reaction material during the thermal cycling.

The materials used to construct the reactors 5 may be plastics, elastomer, glass, metal, ceramic and their combinations, in which the plastics include polypropylene and polycarbonate. The glass reactor 5 can be made in a form of a glass capillary of small diameters such as 0.1 mm-3 mm OD and 0.02 mm-2 mm ID, and the metal can be aluminum in form of thin film, thin cavity, and capillary. Reactor materials can be made from non-biological active substances with chemical or biological stability. At least a portion of the reactor 15 is preferred to be transparent. The volume of the at least one reactor 5 may be in the range 1 μL to 500 μL. Smaller the volume, faster is the heat transfer, higher is the speed of PCR, smaller are the required heating block sizes and more compact is the apparatus. The reaction material in all the reactors 5 in the reactor holder 4 may not be identical. Simultaneous PCR can be advantageously conducted for different materials if the heating block temperatures are suitable. The heating blocks may be shared between different process steps by altering the temperatures. The embodiments described above may be suitable for one reactor 5 or a plurality reactors 5. The reactor 5 may be in the form of tube(s) as shown or as wellplate(s) or chip(s) or cartridge(s) and the like.

When using the above described apparatus for nucleic acid analysis and processing, the reaction material comprises reaction constituents including at least one enzyme, nucleic acid and/or particle containing at least one nucleic acid, primers for PCR, primers for isothermal amplifications, primers for other nucleic acid amplifications and processing, dNTP, Mg²⁺, fluorescent dyes and probes, control DNA, control RNA, control cells, control micro-organisms, and other reagents required for nucleic acid amplification, processing, and analysis. The particle containing nucleic acid mentioned above comprises at least one cell virus, white blood cell and stromal cell, circulating tumor cell, embryo cell. One application may be to use the apparatus to test different kinds of reaction materials against the same set of primer and probes, such as test more than one sample. For such application, different kinds of reaction material containing no target primers and/or probes are each loaded into one reactor 5 in a reactor array, with all the reactors 5 being pre-loaded with the same set or the same sets of PCR primers and/or probes. For the same application, different kinds of reaction materials pre-mixed with respective PCR target primers and/or probes are each loaded into one reactor 5 in a reactor array, with all the reactors 5 being not pre-loaded with the same set of PCR primers and or probes. The reaction materials can include control genes and/or cells and corresponding fluorescent dyes or probes. In the above situations, the different probes emit light of different wavelengths. Another application of the methods and devices are used to test the same reaction material against different sets of primer and probes. One example of such an application is to test one type of sample for more than one purpose. For this application, a single reaction material is added into the reactors 5 each loaded with at least one different set PCR primers and or probes. The reaction material can include control genes and/or cells and corresponding fluorescent dyes or probes. In the above situations, the different probes emit light of different wavelengths. The above reaction material is used in polymerase chain reaction, reverse transcription-PCR, end-point PCR, ligase chain reaction, pre-amplification or target enrichment of nucleic acid sequencing or variations of polymerase chain reaction (PCR), isothermal amplification, linear amplification, library preparations for sequencing, bridge amplification used in sequencing. The variation of the polymerase chain reaction mentioned above comprises reverse transcription-PCR, real-time fluorescent quantitative polymerase chain amplification reaction and real-time fluorescent quantitative reverse transcription polymerase chain amplification reaction, inverse polymerase chain amplification reaction, anchored polymerase chain amplification reaction, asymmetric polymerase chain amplification reaction, multiplex PCR, colour complementation polymerase chain amplification reaction, immune polymerase chain amplification reaction, nested polymerase chain amplification reaction, the target enrichment of pre-amplification or nucleic acid sequencing, ELISA-PCR.

From the foregoing description it will be understood by those skilled in the art that many variations or modifications in details of design, construction and operation may be made without departing from the present invention as defined in the claims. 

What is claimed is:
 1. An apparatus for thermally processing a reaction material containing nucleic acid, the reaction material being contained in a reactor and the reactor being in a form of a tube, a wellplate, a chip, or a cartridge, the apparatus comprising: a reactor holder for statically holding the reactor; at least two heating blocks each being maintainable at a user specifiable temperature; and a transport means for positioning the at least two heating blocks to make a contact with the reactor one at a time for a specified duration, wherein the positioning is conductable once or over a plurality of times for thermally processing the reactor between a plurality of the user specifiable temperatures.
 2. The apparatus according to claim 1, wherein the transport means provides a linear displacement for the at leak two heating blocks.
 3. The apparatus according to claim 2, wherein the transport means further provides a rotational movement for moving the at leak two heating blocks in a circular path, and the rotational movement is about a common axis for the at least two heating blocks.
 4. The apparatus according to claim 3, wherein the circular path is in a vertical plane.
 5. The apparatus according to claim 3, wherein the circular path is in a horizontal plane.
 6. The apparatus according to claim 1, wherein the contact is made when the reactor gets inserted into a plurality of cavities in at least one of the heating blocks, a shape of the plurality of cavities is substantially conformal with at least a lower portion of the reactor.
 7. The apparatus according to claim 1, wherein the at least two heating blocks comprise a thermally conductive elastomeric material.
 8. The apparatus according to claim 7, wherein the thermally conductive elastomeric material is silicone or rubber.
 9. The apparatus according to claim 1, wherein the at least two heating blocks each comprises a porous metal wherein a plurality of voids of the porous metal are filled by a thermally conductive liquid.
 10. The apparatus according to claim 6, wherein the at least two heating blocks each comprises: a first portion coupled to the transport means; and a second portion removably coupled to the first portion, wherein the second portion accommodates the plurality of cavities.
 11. The apparatus according to claim 6, wherein the at least two heating blocks each comprises: at least one injector means, wherein the injector means provides a pressure on the reactor to release the contact in an operation, before the reactor separates out of the at least two heating blocks.
 12. The apparatus according to claim 11, wherein the injector means accesses the reactor via a through hole in each of the at least two heating blocks.
 13. The apparatus according to claim 1, wherein at least one of the at least two heating blocks is a solid metallic block.
 14. The apparatus according to claim 1, wherein at least one of the at least two heating blocks is a hollow metallic block capable of accommodating a heating medium.
 15. The apparatus according to claim 1, further comprising: a fluorescence imaging means, wherein the fluorescence imaging means allows light to access the nucleic acid when the reactor is in contact with any one of the at least two heating blocks or is in air outside the at least two heating blocks.
 16. The apparatus according to claim 1, wherein at least a portion of at least one of the at least two heating blocks is transparent to light for a fluorescent imaging of the reaction material while the reactor is in the contact with the at least one of the at least two heating blocks.
 17. The apparatus according to claim 16, wherein each one of the at least two heating blocks is at least partially made of a transparent material that is in contact with a transparent electrical conductive layer for enabling a resistive heating.
 18. The apparatus according to claim 1 comprising a temperature sensor means or a time sensor means to determine the specified duration.
 19. The apparatus according to claim 18, wherein the temperature sensor means is located to sense a real-time temperature in a sample reactor containing the reaction material.
 20. The apparatus according to claim 18 further comprising a calibration means for a user to calibrate the time sensor means.
 21. The apparatus according to claim 1, wherein the at least two heating blocks each comprises: a first heating block, wherein the reactor is allowed to attain a predetermined high target temperature T_(HT) in the first heating block, the predetermined high target temperature T_(HT) is in a region of 85-99 degree Celsius for a denaturation of the nucleic acid; and a second heating block, wherein the reactor is allowed to attain a predetermined low target temperature T_(LT) in the second heating block, the predetermined low target temperature T_(LT) is in a region of 45-75 degree Celsius for annealing of primers or probes onto the nucleic acid or for a primer extension; wherein, the first and the second heating blocks are for a thermal cycling of the reactor to attain a polymerase chain reaction (PCR) amplification or primer extension.
 22. The apparatus according to claim 1, wherein the reactor is allowed to thermally stabilize in any one of the at least two heating blocks.
 23. The apparatus according to claim 1, wherein at least one of the at least two heating blocks is capable of providing temperatures with programmable ramp up or ramp down characteristics.
 24. The apparatus according to claim 1, wherein the apparatus is configured to provide the user specifiable temperatures at T_(H) and T_(L) such that in the specified duration the reactor alternately attains: a predetermined high target temperature T_(HT), and a predetermined low target temperature T_(LT), while the apparatus adapts to a temperature-offset feature defined by at least one condition selected from the group consisting of: a) the T_(HT) is lower than the T_(H), and b) the T_(LT) is higher than the T_(L).
 25. The apparatus according to claim 24, wherein at least one condition is selectable from the group consisting of: a) the T_(H) is above 100 degrees Celsius, and b) the T_(L) is lower than a room temperature. 